Development of Molecular Contrast-enhanced Imaging for Optical Coherence Tomography

Biological imaging techniques that are able to detect a contrast-enhanced signal from the target molecules have been widely applied to various techniques in the imaging field. The complex biological environment provides numerous and more efficient pathways along which the chromophores (light absorber) may release its energy. This energy can provide not only morphological information, but also specific molecular information such as a biochemical map of a sample. All diseases correlate with both morphological and biochemical changes. Optical coherence tomography (OCT) system is one of the biological imaging techniques. OCT has widely been applied to many medical/clinical fields, giving benefit from a penetration depth of a few millimeters while maintaining a spatial resolution on the order of a micron. Unfortunately, OCT lacks the straightforward functional molecular imaging extensions available for other technologies, e.g. confocal fluorescence microscopy and fluorescence diffuse optical tomography. This is largely because incoherent processes such as fluorescence emission and Raman scattering are not readily detectable with low coherence interferometry that is the central technique that underlies all OCT systems. Despite a drawback of molecular imaging with OCT, it is highly desirable to measure not only morphological, but also molecular information from either endogenous or exogenous molecules. In order to overcome the limitation of molecular contrast imaging for OCT, our group has been researched the hybrid OCT imaging technique and a new exogenous contrast agent. Our contrast-enhanced imaging technique integrates OCT with a well-researched and well-established technique: two-colored pump-probe absorption spectroscopy. Our novel imaging technique is called Pump-Probe OCT (PPOCT). Based upon current successful results, molecular imaging with OCT potentially gives us the ability to identify pathologies. In order to expand the capacity of PPOCT, this dissertation focuses on development of molecular contrast-enhanced imaging for optical coherence tomography (OCT). In the first phase of the research, we developed and optimized for sensitivity a two-color ground state recovery Pump-Probe Optical Coherence Tomography (gsrPPOCT) system and signal algorithm to measure the contrast-enhanced signal of endogenous and exogenous contrast agents such as Hemoglobin (Hb) and Methylene blue (MB) from in vivo samples. Depending on the absorption peak of a target molecule, the pump light sources for PPOCT used 532nm Q-switched laser or 663nm diode laser. Based on different experimental application, Ti:sapp or SLD of 830nm center wavelength were utilized. The PPCOT system was firstly used to image Hb of in vivo vasulature in a Xenopus laevis as the endogenous contrast agent and a larval stage zebrafish using MB as the exogenous contrast agent via transient changes in light absorption. Their morphological in addition to molecular specific information from a live animal was described. The incorporation of a pump laser in an otherwise typical spectrometer based OCT system is sufficient to enable molecular imaging with PPOCT. In the second phase of this research, based on endoscopic molecular contrast-enhanced applications for OCT, we invented an ultra-wideband lensless fiber optic rotary joint based on co-aligning two optical fibers has excellent performance (~0.38 dB insertion loss). The developed rotary joint can cover a wavelength range of at least 355- 1360 nm with single mode, multimode, and double clad fibers with rotational velocities up to 8800 rpm (146 Hz). In the third phase of this research, we developed and manufactured a microencapsulated methylene blue (MB) contrast agent for PPOCT. The poly lactic coglycolic acid (PLGA) microspheres loaded with MB offer several advantages over bare MB. The microsphere encapsulation improves the PPOCT signal both by enhancing the scattering and preventing the reduction of MB to leucomethylene blue. The surface of the microsphere can readily be functionalized to enable active targeting of the contrast agent without modifying the excited state dynamics of MB that enable PPOCT imaging. Both MB and PLGA are used clinically. PLGA is FDA approved and used in drug delivery and tissue engineering applications. 2.5 µm diameter microspheres were synthesized with an inner core containing 0.01% (w/v) aqueous MB. As an initial demonstration the MB microspheres were imaged in a 100 µm diameter capillary tube submerged in a 1% intralipid emulsion. By varying the oxygen concentration both 0% and 21%, we observed he lifetime of excited triple state using time-resolved Pump-Probe spectroscopy and also the relative phase shift between the pump and probe is a reliable indicator of the oxygen concentration. Furthermore, these results are in good agreement with our theoretical predictions. This development opens up the possibility of using MB for 3-D oxygen sensing with PPOCT

Development of Molecular Contrast-enhanced Imaging for Optical Coherence Tomography

Biological imaging techniques that are able to detect a contrast-enhanced signal from the target molecules have been widely applied to various techniques in the imaging field. The complex biological environment provides numerous and more efficient pathways along which the chromophores (light absorber) may release its energy. This energy can provide not only morphological information, but also specific molecular information such as a biochemical map of a sample. All diseases correlate with both morphological and biochemical changes. Optical coherence tomography (OCT) system is one of the biological imaging techniques. OCT has widely been applied to many medical/clinical fields, giving benefit from a penetration depth of a few millimeters while maintaining a spatial resolution on the order of a micron. Unfortunately, OCT lacks the straightforward functional molecular imaging extensions available for other technologies, e.g. confocal fluorescence microscopy and fluorescence diffuse optical tomography. This is largely because incoherent processes such as fluorescence emission and Raman scattering are not readily detectable with low coherence interferometry that is the central technique that underlies all OCT systems. Despite a drawback of molecular imaging with OCT, it is highly desirable to measure not only morphological, but also molecular information from either endogenous or exogenous molecules. In order to overcome the limitation of molecular contrast imaging for OCT, our group has been researched the hybrid OCT imaging technique and a new exogenous contrast agent. Our contrast-enhanced imaging technique integrates OCT with a well-researched and well-established technique: two-colored pump-probe absorption spectroscopy. Our novel imaging technique is called Pump-Probe OCT (PPOCT). Based upon current successful results, molecular imaging with OCT potentially gives us the ability to identify pathologies. In order to expand the capacity of PPOCT, this dissertation focuses on development of molecular contrast-enhanced imaging for optical coherence tomography (OCT). In the first phase of the research, we developed and optimized for sensitivity a two-color ground state recovery Pump-Probe Optical Coherence Tomography (gsrPPOCT) system and signal algorithm to measure the contrast-enhanced signal of endogenous and exogenous contrast agents such as Hemoglobin (Hb) and Methylene blue (MB) from in vivo samples. Depending on the absorption peak of a target molecule, the pump light sources for PPOCT used 532nm Q-switched laser or 663nm diode laser. Based on different experimental application, Ti:sapp or SLD of 830nm center wavelength were utilized. The PPCOT system was firstly used to image Hb of in vivo vasulature in a Xenopus laevis as the endogenous contrast agent and a larval stage zebrafish using MB as the exogenous contrast agent via transient changes in light absorption. Their morphological in addition to molecular specific information from a live animal was described. The incorporation of a pump laser in an otherwise typical spectrometer based OCT system is sufficient to enable molecular imaging with PPOCT. In the second phase of this research, based on endoscopic molecular contrast-enhanced applications for OCT, we invented an ultra-wideband lensless fiber optic rotary joint based on co-aligning two optical fibers has excellent performance (~0.38 dB insertion loss). The developed rotary joint can cover a wavelength range of at least 355- 1360 nm with single mode, multimode, and double clad fibers with rotational velocities up to 8800 rpm (146 Hz). In the third phase of this research, we developed and manufactured a microencapsulated methylene blue (MB) contrast agent for PPOCT. The poly lactic coglycolic acid (PLGA) microspheres loaded with MB offer several advantages over bare MB. The microsphere encapsulation improves the PPOCT signal both by enhancing the scattering and preventing the reduction of MB to leucomethylene blue. The surface of the microsphere can readily be functionalized to enable active targeting of the contrast agent without modifying the excited state dynamics of MB that enable PPOCT imaging. Both MB and PLGA are used clinically. PLGA is FDA approved and used in drug delivery and tissue engineering applications. 2.5 µm diameter microspheres were synthesized with an inner core containing 0.01% (w/v) aqueous MB. As an initial demonstration the MB microspheres were imaged in a 100 µm diameter capillary tube submerged in a 1% intralipid emulsion. By varying the oxygen concentration both 0% and 21%, we observed he lifetime of excited triple state using time-resolved Pump-Probe spectroscopy and also the relative phase shift between the pump and probe is a reliable indicator of the oxygen concentration. Furthermore, these results are in good agreement with our theoretical predictions. This development opens up the possibility of using MB for 3-D oxygen sensing with PPOCT

Enhanced Optical Coupling and Raman Scattering via Microscopic Interface Engineering

Spontaneous Raman scattering is an extremely powerful tool for the remote detection and identification of various chemical materials. However, when those materials are contained within strongly scattering or turbid media, as is the case in many biological and security related systems, the sensitivity and range of Raman signal generation and detection is severely limited. Here, we demonstrate that through microscopic engineering of the optical interface, the optical coupling of light into a turbid material can be substantially enhanced. This improved coupling facilitates the enhancement of the Raman scattering signal generated by molecules within the medium. In particular, we detect at least two-orders of magnitude more spontaneous Raman scattering from a sample when the pump laser light is focused into a microscopic hole in the surface of the sample. Because this approach enhances both the interaction time and interaction region of the laser light within the material, its use will greatly improve the range and sensitivity of many spectroscopic techniques, including Raman scattering and fluorescence emission detection, inside highly scattering environments

Enhanced Coupling of Light into a Turbid Medium through Microscopic Interface Engineering

There are many optical detection and sensing methods used today that provide powerful ways to diagnose, characterize, and study materials. For example, the measurement of spontaneous Raman scattering allows for remote detection and identification of chemicals. Many other optical techniques provide unique solutions to learn about biological, chemical, and even structural systems. However, when these systems exist in a highly scattering or turbid medium, the optical scattering effects reduce the effectiveness of these methods. In this article, we demonstrate a method to engineer the geometry of the optical interface of a turbid medium, thereby drastically enhancing the coupling efficiency of light into the material. This enhanced optical coupling means that light incident on the material will penetrate deeper into (and through) the medium. It also means that light thus injected into the material will have an enhanced interaction time with particles contained within the material. These results show that, by using the multiple scattering of light in a turbid medium, enhanced light-matter interaction can be achieved; this has a direct impact on spectroscopic methods such as Raman scattering and fluorescence detection in highly scattering regimes. Furthermore, the enhanced penetration depth achieved by this method will directly impact optical techniques that have previously been limited by the inability to deposit sufficient amounts of optical energy below or through highly scattering layers

Automated Segmentation of Optical Coherence Tomography Images of the Human Tympanic Membrane Using Deep Learning

Optical Coherence Tomography (OCT) is a light-based imaging modality that is used widely in the diagnosis and management of eye disease, and it is starting to become used to evaluate for ear disease. However, manual image analysis to interpret the anatomical and pathological findings in the images it provides is complicated and time-consuming. To streamline data analysis and image processing, we applied a machine learning algorithm to identify and segment the key anatomical structure of interest for medical diagnostics, the tympanic membrane. Using 3D volumes of the human tympanic membrane, we used thresholding and contour finding to locate a series of objects. We then applied TensorFlow deep learning algorithms to identify the tympanic membrane within the objects using a convolutional neural network. Finally, we reconstructed the 3D volume to selectively display the tympanic membrane. The algorithm was able to correctly identify the tympanic membrane properly with an accuracy of ~98% while removing most of the artifacts within the images, caused by reflections and signal saturations. Thus, the algorithm significantly improved visualization of the tympanic membrane, which was our primary objective. Machine learning approaches, such as this one, will be critical to allowing OCT medical imaging to become a convenient and viable diagnostic tool within the field of otolaryngology

Social vulnerability, parity and food insecurity in urban South African young women: the healthy life trajectories initiative (HeLTI) study.

Social vulnerability indices (SVI) can predict communities vulnerability and resilience to public health threats such as drought, food insecurity or infectious diseases. Parity has yet to be investigated as an indicator of social vulnerability in young women. We adapted an SVI score, previously used by the US Centre for Disease Control (CDC), and calculated SVI for young urban South African women (n = 1584; median age 21.6, IQR 3.6 years). Social vulnerability was more frequently observed in women with children and increased as parity increased. Furthermore, young women classified as socially vulnerable were 2.84 times (95% CI 2.10-3.70; p < 0.001) more likely to report household food insecurity. We collected this information in 2018-2019, prior to the current global COVID-19 pandemic. With South Africa having declared a National State of Disaster in March 2020, early indicators suggest that this group of women have indeed been disproportionally affected, supporting the utility of such measures to inform disaster relief efforts

Social vulnerability, parity and food insecurity in urban South African young women: the healthy life trajectories initiative (HeLTI) study

Social vulnerability indices (SVI) can predict communities’ vulnerability and resilience to public health threats such as drought, food insecurity or infectious diseases. Parity has yet to be investigated as an indicator of social vulnerability in young women. We adapted an SVI score, previously used by the US Centre for Disease Control (CDC), and calculated SVI for young urban South African women (n = 1584; median age 21.6, IQR 3.6 years). Social vulnerability was more frequently observed in women with children and increased as parity increased. Furthermore, young women classified as socially vulnerable were 2.84 times (95% CI 2.10–3.70; p &lt; 0.001) more likely to report household food insecurity. We collected this information in 2018–2019, prior to the current global COVID-19 pandemic. With South Africa having declared a National State of Disaster in March 2020, early indicators suggest that this group of women have indeed been disproportionally affected, supporting the utility of such measures to inform disaster relief efforts.</p